Unité de Virologie Moléculaire et Structurale, Unité Mixte de Recherche, CNRS 2472, Institut Fédératif de Recherche 1157, and IFR 115, Bâtiment 14B, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
J Biol Chem. 2010 Mar 5;285(10):7366-73. doi: 10.1074/jbc.M109.061010. Epub 2010 Jan 7.
DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.
尾部噬菌体和其他病毒的 DNA 包装需要在衣壳顶点组装一个复杂的分子机器。该机器由提供 DNA 进入通道的门户蛋白、为 DNA 易位提供动力的 ATP 酶（大型末端酶亚基）以及最常见的小末端酶亚基组成。在这里，我们研究了噬菌体 SPP1（gp2）的末端酶 ATP 酶亚基与衣壳顶点之间的相互作用。通过用纯化蛋白进行亲和下拉测定，我们发现 gp2 可独立于末端酶小亚基 gp1、DNA 或 ATP 与门户蛋白 gp6 相互作用。通过门户蛋白的 gp2-衣壳相互作用取决于 gp2 浓度，并需要二价阳离子的存在。竞争实验表明，分离的 gp6 只能在 gp6:衣壳摩尔比高于 10 倍时抑制 gp2-衣壳相互作用和 DNA 包装。在其结构的不同区域携带影响 ATP 酶和 DNA 包装的门户诱导刺激的突变的 gp6 的测定显示，这些突变均不阻碍 gp2-gp6 结合。我们的结果表明，SPP1 包装 ATP 酶直接与门户结合，并且与嵌入衣壳中的门户的相互作用更强。鉴定允许 ATP 酶-门户复合物组装但损害 DNA 包装的 gp6 突变支持这两种蛋白之间复杂的交叉对话，以实现 DNA 易位马达的活性。
